title: Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells.
abstract: The aim of this study was to develop a highly specific and sensitive (RT-)PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells. A database of germline immunoglobulin sequences was used to design 112 primers for a nested (RT-)PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. Most but not all V gene family specific primers designed allow amplification of full-length V genes. The nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vkappa families, and all Vlambda genes. The broad V gene family coverage of our primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner. We successfully amplified the expressed VH and Vkappa gene from a single hybridoma cell by RT-PCR and from 10-15 microdissected B cells by genomic PCR. This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Soma
authors.initials: S
authors.lastname: Rohatgi
authors.forename: Parul
authors.initials: P
authors.lastname: Ganju
authors.forename: Devinder
authors.initials: D
authors.lastname: Sehgal
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics* / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D012313:RNA; D004247:DNA